Molecular Weight Phosphotyrosine Protein Phosphatase Research Articles

LMW-PTP modulates glucose metabolism in cancer cells

BackgroundLow Molecular Weight Phosphotyrosine Protein Phosphatase (LMW-PTP) is an enzyme involved not only in tumor onset and progression but also in type 2 diabetes. A recent review shows that LMW-PTP acts on several RTK (receptor tyrosine kinase) such as PDGFR, EGFR, EphA2, Insulin receptor. It is well described also its interaction with cSrc. It is noteworthy that most of these conclusions are based on the use of cell lines expressing low levels of LMW-PTP. The aim of the present study was to discover new LMW-PTP substrates in aggressive human tumors where the over-expression of this phosphatase is a common feature. MethodsWe investigated, by proteomic analysis, the protein phosphorylation pattern of A375 human melanoma cells silenced for LMW-PTP. Two-dimensional electrophoresis (2-DE) analysis, followed by western blot was performed using anti-phosphotyrosine antibodies, in order to identify differentially phosphorylated proteins. ResultsProteomic analysis pointed out that most of the identified proteins belong to the glycolytic metabolism, such as α-enolase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase, suggesting an involvement of LMW-PTP in glucose metabolism. Assessment of lactate production and oxygen consumption demonstrated that LMW-PTP silencing enhances glycolytic flux and slow down the oxidative metabolism. In particular, LMW-PTP expression affects PKM2 tyrosine-phosphorylation and nuclear localization, modulating its activity. ConclusionAll these findings propose that tumor cells are subjected to metabolic reprogramming after LMW-PTP silencing, enhancing glycolytic flux, probably to compensate the inhibition of mitochondrial metabolism. General significanceOur results highlight the involvement of LMW-PTP in regulating glucose metabolism in A375 melanoma cells.

Antitumor activity of irradiated riboflavin on human renal carcinoma cell line 786-O.

Riboflavin (vitamin B2) is a precursor for coenzymes involved in energy production, biosynthesis, detoxification, and electron scavenging. Previously, we demonstrated that irradiated riboflavin (IR) has potential antitumoral effects against human leukemia cells (HL60), human prostate cancer cells (PC3), and mouse melanoma cells (B16F10) through a common mechanism that leads to apoptosis. Hence, we here investigated the effect of IR on 786-O cells, a known model cell line for clear cell renal cell carcinoma (CCRCC), which is characterized by high-risk metastasis and chemotherapy resistance. IR also induced cell death in 786-O cells by apoptosis, which was not prevented by antioxidant agents. IR treatment was characterized by downregulation of Fas ligand (TNF superfamily, member 6)/Fas (TNF receptor superfamily member 6) (FasL/Fas) and tumor necrosis factor receptor superfamily, member 1a (TNFR1)/TNFRSF1A-associated via death domain (TRADD)/TNF receptor-associated factor 2 (TRAF) signaling pathways (the extrinsic apoptosis pathway), while the intrinsic apoptotic pathway was upregulated, as observed by an elevated Bcl-2 associated x protein/B-cell CLL/lymphoma 2 (Bax/Bcl-2) ratio, reduced cellular inhibitor of apoptosis 1 (c-IAP1) expression, and increased expression of apoptosis-inducing factor (AIF). The observed cell death was caspase-dependent as proven by caspase 3 activation and poly(ADP-ribose) polymerase-1 (PARP) cleavage. IR-induced cell death was also associated with downregulation of v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homologue (avian)/protein serine/threonine kinase B/extracellular signal-regulated protein kinase 1/2 (Src/AKT/ERK1/2) pathway and activation of p38 MAP kinase (p38) and Jun-amino-terminal kinase (JNK). Interestingly, IR treatment leads to inhibition of matrix metalloproteinase-2 (MMP-2) activity and reduced expression of renal cancer aggressiveness markers caveolin-1, low molecular weight phosphotyrosine protein phosphatase (LMWPTP), and kinase insert domain receptor(a type III receptor tyrosine kinase) (VEGFR-2). Together, these results show the potential of IR for treating cancer.

Proteomic analysis indicates that overexpression and nuclear translocation of lactoylglutathione lyase (GLO1) is associated with tumor progression in murine fibrosarcoma

Lactoylglutathione lyase (GLO1), a ubiquitously expressed methylglyoxal (MG) detoxification enzyme, is implicated in the progression of various human malignant diseases. However, the role of GLO1 in the development or progression of murine fibrosarcoma is still unclear. We performed proteomic analysis to identify differences in the intracellular proteins of the regressive tumor cell line QR-32 and the inflammatory cell-promoting progressive tumor cell line QRsP-11 of murine fibrosarcoma by 2DE combined with MS. Seven upregulated proteins were identified in QRsP-11 compared to QR-32 cells, namely, GLO1, annexin A1, adenylate kinase isoenzyme 1, transcription factor BTF3, myosin light polypeptide 6, low molecular weight phosphotyrosine protein phosphatase and nucleoside diphosphate kinase B. Heat shock protein beta-1 (HspB1), a methylglyoxal-adducted protein, is concomitantly over-expressed in QRsP-11 as compared to QR-32 cells. We also found out that GLO1 is translocated into the nucleus to a higher extent in QRsP-11 compared to QR-32 cells, which can be reversed by using a MEK inhibitor (U0126). Moreover, U0126 and GLO1 siRNA can inhibit cell proliferation and migration in QRsP-11 cells. Our data suggest that overexpression and nuclear translocation of GLO1 might be associated with tumor progression in murine fibrosarcoma.

Comparative Proteomic Profiling of Human Breast Cell Lines

Although epidemiological studies have suggested that the isoflavone genistein found in soy may be protective against breast cancer, its effects on molecular processes involved in cell proliferation have not been clearly elucidated. Using comparative shotgun proteomics, we examined the proteome of non‐cancerous breast MCF10A cells, estrogen receptor‐positive breast cancer T47D cells, and Hras transfected MCF10A cells after treatment with 3, 10, and 30 μM genistein for 18 h. While genistein induced G1‐phase arrest in MCF10A‐Hras and G2/M‐phase arrest in T47D cells, it did not alter the cell cycle distribution in MCF10A cells. The arrest in G1‐phase of MCF10A‐Hras cells coincided with downregulation of the proliferation marker Ki67, epidermal growth factor receptor, various components of the mitogen‐activated protein kinase family of enzymes and low molecular weight phosphotyrosine protein phosphatase (ACP1), The arrest in G2/M‐phase of T47D cells was accompanied by upregulation of caspase‐8 and mSH2, suggesting that genistein may induce a G2/M checkpoint/proapoptotic response involving the mismatch repair system. Taken together, these findings suggest a differential response to genistein in cancerous cells and non‐malignant epithelial cells, and demonstrate the utility of comparative shotgun proteomics for investigating the cancer preventative effects of bioactive components.

Effects of a Synthetic bFGF Antagonist Peptide on the Proteome of 3T3 Cells Stimulated with bFGF

It has been known that basic fibroblast growth factor (bFGF) plays an important role in tumor progression mainly due to its strong mitogenic activity. Antagonists targeting bFGF have been considered a potential strategy for cancer therapy via inhibiting cell proliferation induced by bFGF. We have previously obtained a high-affinity bFGF-binding peptide (named as P7) with strong inhibitory activity against bFGF-induced cell proliferation from the phage display random heptapeptide library. The aim of the present investigation was to synthesize the peptide P7 by solid phase method and explore the mechanisms of the inhibitory effect of P7-targeting bFGF stimulation on Balb/c 3T3 cells via proteomic analysis. Seven differentially expressed protein were identified, among which four were decreased by bFGF stimulation alone and increased by addition of P7, the other three were up-regulated by bFGF treatment alone and down-regulated by addition of P7. Among the identified proteins, epidermal fatty acid-binding protein (E-FABP) and low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) take part in the regulation of cell proliferation. The results suggested P7 inhibited the bioactivities of bFGF possibly via influencing the expression of cellular proteins related to cell proliferation.

The EphA8 receptor phosphorylates and activates low molecular weight phosphotyrosine protein phosphatase in vitro.

Low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) has been implicated in modulating the EphB1-mediated signaling pathway. In this study, we demonstrated that the EphA8 receptor phosphorylates LMW-PTP in vitro. In addition, we discovered that mixing these two proteins leads to EphA8 dephosphorylation in the absence of phosphatase inhibitors. Finally, we demonstrated that LMW-PTP, modified by the EphA8 autokinase activity, possesses enhanced catalytic activity in vitro. These results suggest that LMW-PTP may also participate in a feedback-control mechanism of the EphA8 receptor autokinase activity in vivo.

Low molecular weight phosphotyrosine protein phosphatase from PC12 cells: Purification, some properties and expression during neurogenesis in vitro and in vivo

The purification and partial characterization of low molecular weight phosphotyrosine phosphatase (LMW-PTP) was reported for the first time in PC12 cells. In addition, the expression levels during neuronal phenotype induction by nerve growth factor (NGF) and during neurogenesis in chick embryos were investigated. LMW-PTP was purified to homogeneity and showed a single band of about 18 kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis. A native molecular mass of 20.1 kDa was determined by gel filtration on Sephadex G-75 column. The LMW-PTP from PC12 cells displays structural and biochemical characteristics similar to the enzyme isolated for normal tissues. It was specifically immunoprecipitated by an affinity purified antibody directed against the bovine liver enzyme. The enzyme is present in the cytosolic and cytoskeletal cell compartment where is tyrosine phosphorylated. Time course expression of LMW-PTP in PC12 cells was investigated after NGF treatment and showed an increase of about 30% in the basal level of LMW-PTP from 0 to 72 h. These changes were related to the appearance in PC12 cells of neuronal processes and to a decrease in cell proliferation. An increase of the LMW-PTP expression was also observed in vivo during chick embryo neurogenesis from 8-day-old embryos to adult chicks. The protein level, assayed by immunoblotting, increases from 14-day-old embryos to the hatched chicks reaching the adult levels within the first week after birth. These data indicate that the neurogenesis process is accompanied by a physiological increment of LMW-PTP expression in vitro and in vivo.

Acid phosphatase locus 1 (ACP1): Possible relationship of allelic variation to body size and human population adaptation to thermal stress-A theoretical perspective.

The acid phosphatase locus 1 (ACP1) codes for a low molecular weight phosphotyrosine protein phosphatase that has the important action of dephosphorylating tyrosine phosphorylated proteins and peptides and a second important role in modulating flavin cofactor levels and the activity of flavo-enzymes. These functions significantly influence cell division, differentiation, and growth. Two alleles (ACP1*A and ACP1*B) reach polymorphic frequencies at the ACP1 locus in all human populations, while the ACP1*C and ACP1*R alleles reach polymorphic frequencies in restricted geographical regions. The worldwide distribution of these alleles, and data from several clinical studies, strongly suggest that the ACP1 locus functions to modulate growth and that selection at this locus is a component of the selective processes influencing body mass and human population adaptation to thermal stress. The ACP1*A allele reaches highest frequencies at extreme latitudes and appears to be associated with maximizing body mass and adaptation to cold stress, whereas the ACP1*B allele reaches highest frequencies in tropical and subtropical environments and appears to be associated with minimizing body mass and adaptation to heat stress. The high frequency of the ACP1*C allele at northern latitudes, where ACP1*A allele frequencies are elevated, may be a mechanism for limiting fetal and maternal complications associated with fetal macrosomia and adult obesity in populations where protein and calorie intake are relatively high. Am. J. Hum. Biol. 12:688-701, 2000. Copyright 2000 Wiley-Liss, Inc.

The low Mr phosphotyrosine protein phosphatase behaves differently when phosphorylated at Tyr 131 or Tyr 132 by Src kinase

The low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) is phosphorylated by Src and Src-related kinases both in vitro and in vivo; in Jurkat cells, and in NIH-3T3 cells, it becomes tyrosine-phosphorylated upon stimulation by PDGF. In this study we show that pp60 Src phosphorylates in vitro the enzyme at two tyrosine residues, Tyr 131 and Tyr 132, previously indicated as the main phosphorylation sites of the enzyme, whereas phosphorylation by the PDGF-R kinase is much less effective and not specific. The effects of LMW-PTP phosphorylation at each tyrosine residue were investigated by using Tyr 131 and Tyr 132 mutants. We found that the phosphorylation at either residue has differing effects on the enzyme behaviour: Tyr 131 phosphorylation is followed by a strong (about 25-fold) increase of the enzyme specific activity, whereas phosphorylation at Tyr 132 leads to Grb2 recruitment. These differing effects are discussed on the light of the enzyme structure.

Abundance of low molecular weight phosphotyrosine protein phosphatase in the nerve-ending fraction in the brain.

The distribution of low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) in subcellular fractions of rat brain tissue was investigated by immunoblotting analysis using anti-LMW-PTP antibody. The enzyme was detected in the 105000 g precipitate in addition to the supernatant of brain homogenate, even after the precipitate was extensively washed, and was abundant in the particulate fraction of nerve endings. Nerve ending LMW-PTP was effectively solubilized by 1% Triton X-100 or 1% deoxycholate, though the enzyme was solubilized by thorough sonication. Two forms of LMW-PTP, designated as LMW-PTP-I and -II, were separated from the nerve ending-rich fraction by chromatofocusing. Nerve endings PTP-I and -II were different in molecular weight, isoelectric point and susceptibility to activators and inhibitors. The properties of nerve endings LMW-PTP-I and -II were similar to those of cytosolic LMW-PTP-I and -II. The abundance of LMW-PTP in nerve endings as well as in the cytosol suggests that this enzyme plays an important role in synaptic function.

Cloning of murine low molecular weight phosphotyrosine protein phosphatase cDNA: identification of a new isoform

The low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) is a 18 kDa cytosolic enzyme, involved in the negative regulation of cell proliferation. In different mammalian species LMW-PTPs are expressed in two molecular forms produced from a single primary transcript through an alternative splicing mechanism. In this paper we report the cloning, expression and characterization of mouse isoforms of LMW-PTPs (called m-IF1 and m-IF2), very similar to the corresponding rat and human isoenzymes. Moreover we have identified a third cDNA encoding a protein (m-IF2P) that presents three substitutions compared to m-IF2. This new isoform is still active on pNPP, although to a lower extent: this reduction is mainly due to the leucine to proline substitution in position 13, within the catalytic loop. The mRNA expression level of this isoform is comparable to those of m-IF1 and m-IF2. It is likely that a gene duplication process followed by mutations has generated this new gene.

Low molecular weight phosphotyrosine protein phosphatase translocation during cell stimulation with platelet-derived growth factor

Low M r phosphotyrosine protein phosphatase (PTP) is a cytosolic enzyme whose activity upon platelet-derived growth factor (PDGF) and insulin receptors has been demonstrated in vivo. In our study we demonstrate that this enzyme, both naturally expressed and overexpressed in NIH/3T3 fibroblasts, translocates from the cytosol to the Triton X-100 insoluble fraction following stimulation with PDGF. It emerges that the phosphorylation of a defined population of PDGF receptors, which is localized in this fraction and seems to be endowed with peculiar features and functions, is particularly affected by low M r PTP overexpression.

Nitric oxide causes inactivation of the low molecular weight phosphotyrosine protein phosphatase.

The low M(r) phosphotyrosine protein phosphatase (PTPase) and Yersinia enterocolitica PTPase are inactivated by nitric oxide-generating compounds. Inorganic phosphate, a competitive inhibitor, protects the enzymes from inactivation, suggesting that the action of NO is directed to the active sites. Low M(r) PTPase from bovine liver lost two out of eight thiol groups present in the molecule during the inactivation with sodium nitroprusside and with other NO-producing compounds. The mass spectrometric analyses of tryptic fragments of the enzyme, performed after chemical modification of the NO-unreacted thiol groups, demonstrated that NO caused the oxidation of Cys-12 and Cys-17 to form an S-S bond. A similar reaction was described previously for the reaction of NO with N-methyl-D-aspartate receptor. The NO-inactivated low M(r) PTPase was reactivated by treating the inactive enzyme with thiol-containing reagents. Since all members of the PTPase family have the same reaction mechanism and possess a conserved active site motif that contains an essential cysteine residue, the findings on low M(r) and Yersinia PTPases are potentially interesting for all PTPases, an enzyme class that is involved in a number of important biological processes.

Crystallisation of a low molecular weight phosphotyrosine protein phosphatase from bovine liver

Single crystals of a low molecular weight phosphotyrosine protein phosphatase from bovine liver have been grown. The crystals belong to space group P2 12 12 1, have cell dimensions a = 46.3 Å, b - 62.2 Å, c = 62.7 Å and diffract to better than 2.0 Å resolution. The crystals are well suited for high resolution X-ray studies.